Apoptosis Might Contribute to the Thrombocytopenia in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a prothrombotic autoimmune complication of heparin therapy. Thrombocytopenia and thrombosis in HIT patients are caused by immune complexes containing pathogenic antibodies against platelet factor 4 (PF4)/glycosaminoglycan complexes. Mechanisms of platelet activation and/or destruction in HIT are not fully understood. Phosphatidylserine expression is a marker of platelet activation that contributes to the procoagulant function. On the other hand, phosphatidylserine expression is generally an early marker of cell apoptosis, which, similarly to other cells, controls platelet life span. The aim of this study was to investigate if apoptosis might play a role in HIT.

Gel-filtered normal platelets were incubated for 15 or 60 min with recombinant PF4 (10 µg/ml) and KKO antibodies (50 µg/ml) and studied by electron microscopy and flow cytometry using fluorescently labeled markers of cell activation and apoptosis, such as annexin V, antibodies to CD62P (P-selectin) and MitoTracker DeepRed FM. Platelets and platelet-derived microparticles were identified by flow cytometry using labeled antibodies to CD41 and electron microscopy. Calcium ionophore A23187 (10 µM) was used as a positive control.

Incubation of platelets with PF4+KKO caused fast expression of P-selectin on platelets comparable with calcium ionophore A23187 stimulation, suggesting that platelets were fully activated by PF4+KKO within 15 min, when they also started to produce CD41 and annexin-positive microparticles. Activation of platelets with PF4+KKO for 60 minutes led to a further increase in phosphatidylserine expression on their surface, with a time-dependent reduction of mitochondrial membrane potential, which reflects a disturbance of energy metabolism and is characteristic of cell apoptosis. Scanning electron microscopy showed that platelets treated with PF4+KKO or A23187, unlike untreated cells, displayed dramatic morphological changes with a loss of discoid shape, formation of filopodia, and microvesiculation. By transmission electron microscopy, the PF4+KKO-treated platelets had an irregular shape due to formation of plasma membrane invaginations and pseudopodia. Formation of an increasing number of intracellular vacuoles and enlargement of the lumen of the open canalicular system were observed. Some vacuoles contained various inclusions, such as secretory granules, membrane components, and grainy particles. The number of secretory granules in the PF4+KKO-treated cells was dramatically reduced. In all cases, formation of microparticles of various shapes and sizes was observed.

These results indicate that the PF4-containing pathogenic immune complexes induce strong and time-dependent platelet activation leading to procoagulant microparticle formation that may contribute to thrombosis. At the same time, the results strongly suggest that the HIT-like immune complexes likely induce platelet apoptosis that can be an important mechanism of thrombocytopenia.